The human pathogen Chlamydia trachomatis is a significant concern in the United States due to its prevalence and the combined health and socioeconomic impact of acute and chronic disease. Chlamydiae are obligate intracellular pathogens and possess the ability to modulate host-cell functions from within a membrane-bound vacuole termed an inclusion. Chlamydiae express a virulence-associated type III secretion system presumably employed to create and maintain a permissive growth environment. We have identified multiple chlamydial type III substrates and propose to elucidate the molecular mechanisms of their anti-host activities and delineate the consequences of those interactions. We, furthermore, propose to extend the number of identified host-interactive chlamydial products using genetic and biochemical approaches. A combination of methods designed to identify interactions of chlamydial proteins with host targets will be employed to establish relevant functions. These elucidated in vitro interactions will be confirmed in chlamydial infections using a series of crosslinking and co-precipitation assays in combination with mass spectroscopy. The consequences of these interactions will be investigated in a tissue culture infection model in order to evaluate the requirement of respective functions in chlamydial development. The chlamydial type III secretion system represents an attractive, yet relatively unexplored, mechanism to achieve modulation of host cell activities. Given the comparative difficulty associated with study of obligate intracellular bacteria, investigation of host pathways specifically targeted by the type III secretion mechanism represents a productive approach to elucidate novel pathogenic mechanisms. These studies will lead to an enhanced understanding of Chlamydia-mediated disease and have the potential to yield novel preventative and treatment therapies.